Cells die throughout the lifespan of multicellular organisms, and the correct regulation of cell death is critical for organismal health, from the earliest stages of development through adult life. We propose to bring together the complementary expertise of two labs whose research has focused on the regulation of cell death in vivo, using the Drosophila model system. These labs bring unique tools and novel approaches to study the complex activation of the Drosophila cell death regulators. These regulators: reaper, hid, grim and sickle, are clustered together in a gene complex. Remarkably, the regulatory regions in this complex are more highly conserved, and are functionally more important, than the individual genes. In this proposal, we focus on transcriptional, epigenetic and conformational changes in this genomic region that are required to initiate cell death. We have developed new tools and approaches to determine how the input from developmental pathways is integrated to regulate cell death, and how chromatin conformation contributes to the initiation of cell death. We will test the hypothesis that changes in chromatin conformation ensure robust expression of pro- apoptotic genes in cells fated to die, while strongly blocking expression in cells destined to live. We will examine the role of three dimensional chromatin interactions in this rapid and precise regulation of specific combinations of genes in response to different intrinsic and extrinsic signals. We will also determine whether feedback from the cell death pathway is important for the maintenance of cell death gene activation. Extensive preliminary data indicate that these studies will be successful. In a large-scale genetic screen, we have identified genes that are required for the physiologic death of a precise subset of cells. These include sequence specific transcriptional regulators, and genes important for chromatin conformation and three dimensional chromosome architectures. We will investigate how these genes activate the cell death gene complex. At the conclusion of this grant, we will have developed a functionally validated model of the control of physiologic cell death. In addition, we will have described general principles for the regulation of gene complexes and shared regulatory regions.